Protein sequencing methods and reagents

ABSTRACT

Described are optical methods and reagents for sequencing polypeptides. A probe that exhibits different spectral properties when conjugated to different N-terminal amino acids is conjugated to the N-terminal amino acid of a polypeptide. Sequentially detecting one or more spectral properties of the probe conjugated to the N-terminal amino acid and cleaving the N-terminal amino acid produces sequence information of the polypeptide. The use of super-resolution microscopy allows for the massively parallel sequencing of individual polypeptide molecules in situ such as within a cell. Also described are probes comprising hydroxymethyl rhodamine green, an isothiocyanate group and a protecting group.

RELATED APPLICATIONS

This application claims the benefit of priority of U.S. ProvisionalPatent Application No. 62/242,619 filed Oct. 16, 2015, the contents ofwhich are hereby incorporated by reference.

FIELD OF THE INVENTION

This invention relates to the field of protein sequencing and morespecifically to methods, assays and reagents for sequencing protein orpolypeptide molecules as well as to fluorescent single molecule imagingmethods, assays and reagents for sequencing individual polypeptidemolecules.

BACKGROUND OF THE INVENTION

Proteins underlie virtually every biological process; perturbations intheir expression, degradation, interactions, or localization areassociated with disease. Yet while methods for protein identification,quantification and imaging are needed throughout biomedicine, existingtechniques have serious constraints.

Historically (1960-90s), protein sequencing was based on Edmandegradation, which involves the sequential chemical modification of theN-terminal amino acid from an immobilized polypeptide population with areactive isothiocyanate-based reporter reagent and their subsequentcleavage and detection by differential chromatography. Whileautomatable, the technology has low sensitivity and is not applicable toheterogeneous samples (i.e. mixtures of different polypeptides),limiting utility. Over the last 20 years, mass spectrometry has emergedas a preferred technology for identifying proteins in biologicalspecimens (i.e. complex mixtures). Yet it also suffers from limiteddynamic range, biased detection, and incremental performance gains.Moreover, it is based on ensemble measurements (bulk analysis),resulting in the loss of valuable contextual information (e.g. proteinsubcellular localization etc.).

Like mass spectrometry, fluorescent microscopy has a long anddistinguished track record in the analyses of cellular proteins.Historically, while spatial resolution was limited by the wavelength oflight, recently introduced super-resolution microscopy techniques basedon single-molecule localization have now overcome fundamentaldiffraction limits. Some of these new methods, like STORM (stochasticoptical reconstruction microscopy), involve switching a sparse subset offluorescent molecules on and off (dark vs. activated states) followed byimage acquisition to allow for very precise probe localization (<20 nmin lateral plane). Crucially, however, these methods depend on theavailability of fluorescent antibodies, or other similarly labeledaffinity capture reagents such as aptamers, which are physically largerelative to most cellular polypeptides, diminishing resolution, and/orwhich may bind cellular proteins in addition to the target of interest,producing artifacts, and typically fewer than 3-4 different cellularproteins can be imaged together at one time.

There remains a need for novel methods and assays for sequencing singlepolypeptide molecules and for identification, quantification and imagingof many different proteins simultaneously in complex biological samples.

SUMMARY OF THE INVENTION

In a broad aspect, the disclosure provides a method for sequencing apolypeptide wherein the N-terminal amino acid of the polypeptide isconjugated to a probe that exhibits different spectral properties whenconjugated to different N-terminal amino acids. In one embodiment, theprobe is an optically active reporter probe, such as a fluorescent dye.

As set out in the Examples, small but characteristic differences inspectral properties observed after conjugation of a probe to differentamino acids may be used to infer the identity of the correspondingN-terminal amino acid residue. Furthermore, the use of single moleculeimaging techniques that allow for the detection of spectral propertiesfor spatially resolved individual molecules, such as but not limited tosuper-resolution microscopy, may be used for the parallel sequencing oflarge numbers of polypeptides in vitro or in situ, such as on or intissues, cells, lipid membranes or organelles. The methods describedherein can therefore also be used to obtain information on proteinidentity, quantity and subcellular location within a biological orenvironmental sample.

Accordingly, in one embodiment there is provided a method of sequencinga polypeptide comprising:

a) conjugating a probe to an N-terminal amino acid of the polypeptidewherein the probe exhibits different spectral properties when conjugatedto different N-terminal amino acids;

b) detecting one or more spectral properties of the probe conjugated tothe N-terminal amino acid;

c) identifying the corresponding N-terminal amino acid of thepolypeptide by comparing the spectral properties of the probe to aplurality of reference spectral properties, wherein each referencespectral property is representative of the probe conjugated to adifferent N-terminal amino acid;

d) cleaving the N-terminal amino acid of the polypeptide; and

e) repeating steps (a) to (d) to determine the sequence of at least aportion of the polypeptide.

In one embodiment, the polypeptide is a single polypeptide molecule.

In one embodiment, the probe is covalently conjugated to the N-terminalamino acid of the polypeptide.

In one embodiment, the probe is an optical reporter probe, such as afluorescent dye. In one embodiment, detecting one or more spectralproperties of the probe conjugated to the N-terminal amino acidcomprises detecting the fluorescence emission of the probe bound to theN-terminal amino acid of the polypeptide at one or a plurality ofwavelengths. In one embodiment, detecting one or more spectralproperties of the probe conjugated to the N-terminal amino acidcomprises detecting an emission intensity, polarity/anisotropy orlifetime. In one embodiment, the probe comprises a fluorescent moiety.In one embodiment, the fluorescent moiety is a xanthene derivative, suchas a dye based on fluorescein, eosin, or rhodamine. In one embodiment,the probe comprises rhodamine green, or a derivative thereof. In oneembodiment, the probe is hydroxymethyl rhodamine green (HMRG)-BOC-ITC).

In one embodiment, the probe comprises a spontaneously blinking dye or aphotoswitchable dye. In one embodiment, the probe comprises areactive/labile isothiocyanate group.

In one embodiment, the N-terminal amino acid of the polypeptide orN-terminal amino acid derivative of the polypeptide is cleaved usingEdman chemical degradation.

In one embodiment, methods described herein can be used to sequence apolypeptide in situ. For example in one embodiment the polypeptide is inor on a biological sample, such as a tissue, cell, lipid membrane orintracellular organelle, or sample thereof. In another embodiment, thepolypeptide is conjugated to substrate prior to conjugating the probe tothe N-terminal amino acid. In one embodiment, the C-terminal end of thepolypeptide is conjugated to a substrate, optionally through a linker.

In one aspect, the methods described herein may be used to sequence aplurality of polypeptides in parallel. For example, in one embodiment,the method comprises conjugating a plurality of probes to the N-terminalamino acid of each of the plurality of polypeptides, detecting one ormore spectral properties for each probe conjugated to the N-terminalamino acid of each of the plurality of polypeptides, and identifying theN-terminal amino acid of each of the plurality of polypeptides bycomparing the plurality of spectra properties to a plurality ofreference (standard) spectral properties.

In one embodiment, the methods described herein comprise detecting oneor more spectral properties for each probe conjugated to the N-terminalamino acid of each of the plurality of polypeptides at spatiallyresolved locations in a sample containing the plurality of polypeptides.For example, in one embodiment, the methods described herein includedetecting one or more spectral properties of a probe using superresolution microscopy, optionally stochastic optical reconstructionmicroscopy (STORM) or related/derivative methods.

In one embodiment, the methods described herein include obtaining afluorescence emission spectra of the probe conjugated to the N-terminalamino acid and comparing the fluorescence emission spectra to aplurality of reference spectra, wherein each reference spectra isrepresentative of the probe conjugated to a different N-terminal aminoacid.

In one embodiment, the method comprises comparing the spectralproperties of the probe conjugated to an N-terminal amino acid to one ora plurality of reference spectral properties. In one embodiment, eachreference spectral property is representative of the probe conjugated toa different N-terminal amino acid. In one embodiment, comparing thespectral properties of the probe to a plurality of reference spectralproperties comprises the use of machine learning, genetic algorithms, orprinciple component analysis (PCA).

The methods described herein may be used in combination with availablepolypeptide sequence information or databases in order to predict thesequence of a polypeptide. For example, in one embodiment the methodcomprises comparing the sequence of at least one polypeptide moleculedetermined using a probe as described herein to a reference proteinsequence database.

Also provided are reagents and probes such as fluorescent opticalreporter probes useful for the method of sequencing a polypeptide asdescribed herein. In one embodiment, the probe exhibits differentspectral properties when conjugated to different N-terminal amino acids,such as different emission intensity, polarity/anisotropy or lifetime.In one embodiment, the probe exhibits a different spectral shape whenconjugated to different N-terminal amino acids.

In one embodiment, the probe comprises a synthetic fluorescent dye. Inone embodiment, the probe comprises a xanthene derivative, such as a dyebased on fluorescein, eosin, or rhodamine. For example, in oneembodiment, the probe comprises hydroxymethyl rhodamine green (HMRG). Inone embodiment, the probe is suitable for optical detection usingsuper-resolution microscopy. In one embodiment, the probe is aspontaneously blinking dye or a photoswitchable dye. In one embodiment,the probe is facilitates the chemical cleavage of the N-terminal aminoacid from the polypeptide. For example, in some embodiment the probecomprises a reactive isothiocyanate (ITC) group.

In one embodiment, there is provided a chemical compound comprising areactive ITC group, HMRG and a protecting group (PG) such astert-Butyloxycarbonyl (BOC).

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from the detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

Further features and advantages of the present invention will becomeapparent from the following detailed description, taken in combinationwith the appended drawings, in which:

FIG. 1 shows a representative frame of the single molecule imaging(STORM) of HMRG coupled, via an ITC moiety, to Immunoglobulin (IgG). Thenumber of localizations recorded per 1000 frames was 573,052. Theaverage fluorescent intensity measured per individual molecule was9111.153 and the average location uncertainty was 18.39 nm.

FIG. 2 shows the different fluorescence emission profiles of HMRGcoupled to Methionine (Met) vs. Tryptophan (Trp) in solution at pH 5.

FIG. 3 shows the different fluorescence emission profiles of HMRGcoupled to Tyrosine (Tyr) vs. Leucine (Leu) in solution at pH 5.

FIG. 4A shows the chemical structure of the probe HMRG-BOC-ITC. FIG. 4Bshows a synthetic scheme for generating HMRG-Boc-ITC.

FIG. 5 shows the spectra of HMRG-BOC-ITC conjugated to a testpolypeptide XAGWYMRLG (SEQ ID NO: 1; wherein X is any amino acid) havingdifferent N-terminal amino acids. The spectra of the polypeptides havingdifferent N-terminal amino acids are readily distinguished.

FIG. 6 shows that the spectra of HMRG-BOC-ITC coupled to hydrophobicN-terminal amino acids (such as Leucine) generally exhibit a red-shiftrelative to the average spectra of each of the 20 amino acids tested.The spectra of HMRG-BOC-ITC coupled to hydrophilic N-terminal aminoacids (such as Aspartic Acid) generally exhibit a blue-shift relative tothe average spectra of each of the 20 amino acids tested.

DETAILED DESCRIPTION OF THE INVENTION

The present description provides molecular imaging-based methods, assaysand reagents useful for sequencing proteins. In one aspect, the methodsand reagents are useful for sequencing single polypeptide molecules,multiple molecules of a single polypeptide, or multiple different singlepolypeptide molecules. In one aspect, the methods and reagents areuseful for determining the N-terminal amino acid of one or morepolypeptides. In one aspect, the methods are useful for the simultaneoussequencing of a plurality of polypeptide molecules, such as formassively parallel sequencing techniques. Accordingly samples comprisinga mixture of different proteins, or peptides, can be assayed accordingto the methods described herein to generate (partial or complete)sequence information regarding individual protein molecules in thesample. In a further aspect, the methods are useful for proteinexpression profiling in biological samples containing complex proteinmixtures such as cells. For example, the methods are useful forgenerating both quantitative (frequency) and qualitative (sequence) datafor proteins contained in a sample. In addition, the methods andreagents described herein are useful for generating data on the locationand/or distribution of proteins within a sample, such as biologicalsample or environmental sample.

The inventor has determined that differences in the spectral propertiesof a probe conjugated to the N-terminal amino acid residue of apolypeptide can be used to determine the identity of the 20+ naturallyoccurring N-terminal amino acid residues. As shown in Example 3 and FIG.5, the spectra of a xanthene-based dye (HMRG-BOC) conjugated todifferent N-terminal amino acids of a test polypeptide (SEQ ID NO: 1)exhibited different spectral signatures allowing for the identificationof the N-terminal amino acid of a polypeptide based on the spectra. Inone embodiment, the methods and reagents described herein can be used togenerate sequence information by sequentially identifying and thencleaving off the N-terminal amino acid of a polypeptide.

Accordingly, in one aspect there is provided a method of sequencing apolypeptide comprising conjugating a probe to an N-terminal amino acidof the polypeptide wherein the probe exhibits different spectralproperties when conjugated to different N-terminal amino acids. In oneembodiment, the probe is an optical reporter probe such as a fluorescentdye. In one embodiment, the method comprises detecting one or morespectral properties of the probe conjugated to the N-terminal aminoacid. In one embodiment, the method comprises identifying the N-terminalamino acid of the polypeptide by comparing the spectral properties ofthe probe to a plurality of reference spectral properties, wherein eachreference spectral property is representative of the probe conjugated toa different N-terminal amino acid. In one embodiment, the methodcomprises repeatedly cleaving off the N-terminal amino acid of thepolypeptide, either chemically or enzymatically, then conjugating thenewly exposed N-terminal amino acid to the probe and detecting one ormore spectral properties of the conjugated probe in order tosequentially identify the consecutive amino acid sequence of thepolypeptide.

As used herein, “polypeptide” refers to two or more amino acids linkedtogether by a peptide bond. The term “polypeptide” includes proteins, orprotein digests, that have a C-terminal end and an N-terminal end asgenerally known in the art and may be synthetic in origin or naturallyoccurring. As used herein “at least a portion of the polypeptide” refersto 2 or more amino acids of the polypeptide. Optionally, a portion ofthe polypeptide includes at least: 5, 10, 20, 30 or 50 amino acids,either consecutive or with gaps, of the complete amino acid sequence ofthe polypeptide, or the full amino acid sequence of the polypeptide.

The phrase “N-terminal amino acid” refers to an amino acid that has afree amine group and is only linked to one other amino acid by a peptideamide bond in the polypeptide. Optionally, the “N-terminal amino acid”may be an “N-terminal amino acid derivative”. As used herein, an“N-terminal amino acid derivative” refers to an N-terminal amino acidresidue that has been chemically modified, for example by an Edmanreagent or other chemical in vitro or inside a cell via a naturalpost-translational modification (e.g. phosphorylation) mechanism.

As used herein, “sequencing a polypeptide” refers to determining theamino acid sequence of a polypeptide. The term also refers todetermining the sequence of a segment of a polypeptide or determiningpartial sequence information for a polypeptide.

As used herein “the cleaving the N-terminal amino acid of thepolypeptide” refers to a chemical or enzymatic reaction whereby theN-terminal amino acid or N-terminal amino acid derivative is removedfrom the polypeptide while the remainder of the polypeptide remainsintact.

As used herein the term “sample” includes any material that contains oneor more polypeptides. The sample may be a biological sample, such asanimal or plant tissues, biopsies, organs, cells, membrane vesicles,plasma membranes, organelles, cell extracts, secretions, urine ormucous, tissue extracts or other biological specimens both natural orsynthetic in origin. The term sample also includes single cells,organelles or intracellular materials isolated from a biologicalspecimen, or viruses, bacteria, fungus or isolates therefrom. The samplemay also be an environmental sample, such as a water sample or soilsample, or a sample of any artificial or natural material that containsone or more polypeptides.

Without being limited by theory, it is believed that the atomicinteractions induced by different amino acid side-chains affect theelectronic (ground or activation) states of the probe conjugated to aparticular amino acid residue. In one embodiment, the probe iscovalently conjugated to the N-terminal amino acid of the polypeptide.

In one embodiment, these changes to the electronic (ground oractivation) state of the probe conjugated to the N-terminal acid residueare detected by detecting changes to one or more spectral properties ofthe probe conjugated to the N-terminal amino acid residue, such asemission intensity, polarity/anisotropy or lifetime. As used herein, theterm “spectral properties” refers to a detectable change in the emissionintensity, polarity/anisotropy or lifetime at a single wavelength or ata plurality of wavelengths of a probe conjugated to an N-terminal aminoacid relative to one or more different N-terminal amino acids. Forexample, in one embodiment spectral properties may include spectralshape or peak intensity and/or polarity. In one embodiment, the methodsdescribed herein include detecting fluorescence of the probe bound tothe N-terminal amino acid of the polypeptide. As shown in FIG. 5, thefluorescent spectra of the xanthene-based probe HMRG-BOC conjugated todifferent N-terminal amino acids of a test polypeptide exhibiteddistinctive spectral properties. Comparing the spectra of can thereforebe used to identify the N-terminal amino acid to which the probe isconjugated. As shown in FIG. 6, HMRG-BOC conjugated to hydrophobicN-terminal amino acids tends to exhibit a red-shift in fluorescentintensity compared to the average of all 20 amino acids while HMRG-BOCconjugated to hydrophilic N-terminal amino acids tends to exhibit ablue-shift in fluorescent intensity compared to the average of all 20amino acids. Optionally, the methods described herein include comparingmultiple spectral properties (e.g. spectral shape and polarity) betweenan N-terminal amino acid-probe conjugate and one or more controls inorder to identify the identity of the N-terminal amino acid.

In one embodiment, the methods described herein comprise detecting thefluorescence emission intensity, polarity/anisotropy or lifetime at asingle wavelength or at a plurality of wavelengths.

For example, in one embodiment, the methods described herein includedetecting a fluorescent emission spectra for a probe conjugated to anamino acid residue, such as those shown in FIG. 2 or 3.

In one embodiment, the probes described herein exhibit differentspectral properties when conjugated to different N-terminal amino acids.For example, various fluorescent dyes known in the art may be tested toidentity those that exhibit unique spectral properties when conjugatedto different amino acid residues.

In one embodiment, the probe comprises a xanthene derivative. Forexample, in one embodiment the probe is a fluorescein, eosin, orrhodamine derivative. For instance, in one embodiment, the fluorescentprobe is a derivative of hydroxymethyl rhodamine green (HMRG). As shownin the Examples and FIG. 2, HMRG exhibits different spectral propertieswhen conjugated to either Methionine or Tryptophan (or other aminoacids). Similarly, FIG. 3 demonstrates different spectral properties ofHMRG conjugated to Leucine or Tryptophan.

In one embodiment, the probe comprises an organic dye suitable for usewith single molecule optical detection techniques. For example, in oneembodiment the probe comprises a spontaneously blinking dye or aphotoswtichable dye.

In one embodiment, the probe facilitates cleavage of the N-terminalamino acid from the polypeptide. For example, in one embodiment theprobe comprises a reactive and labile isothiocyanate group.

In one embodiment, there is provided a compound comprising ahydroxymethyl rhodamine green (HMRG) derivative, a labile isothiocyanategroup and a protecting group (PG) protecting the amine group on theopposite side of the xanthene moiety. In one embodiment, the compoundhas the formula:

The term “protecting group” or “PG” as used herein refers to a chemicalmoiety which protects or masks a reactive portion of a molecule toprevent side reactions in that reactive portion of the molecule, whilemanipulating or reacting a different portion of the molecule. After themanipulation or reaction is complete, the protecting group is removedunder conditions that do not degrade or decompose the remaining portionsof the molecule; i.e. the protected reactive portion of the molecule is“deprotected”. The selection of a suitable protecting group can be madeby a person skilled in the art. Many conventional protecting groups areknown in the art, for example as described in “Protective Groups inOrganic Chemistry” McOmie, J. F. W. Ed., Plenum Press, 1973, in Greene,T. W. and Wuts, P.G.M., “Protective Groups in Organic Synthesis”, JohnWiley & Sons, 3^(rd) Edition, 1999 and in Kocienski, P. ProtectingGroups, 3rd Edition, 2003, Georg Thieme Verlag (The Americas). Examplesof protecting groups include, but are not limited to t-Boc, C₁₋₆acyl,Ac, Ts, Ms, silyl ethers such as TMS, TBDMS, TBDPS, Tf, Ns, Bn, Fmoc,dimethoxytrityl, methoxyethoxymethyl ether, methoxymethyl ether,pivaloyl, p-methyoxybenzyl ether, tetrahydropyranyl, trityl, ethoxyethylethers, carbobenzyloxy, benzoyl and the like. In one embodiment, theprotecting group is an amine protecting group.

In one embodiment, the compound is HMRG-BOC-ITC as shown in FIG. 4A. Anexemplary scheme for synthesizing HMRG-BOC-ITC is shown in FIG. 4B. Alsoprovided is a method for synthesizing the compound HMRG-BOC-ITC as shownin FIG. 4B.

Optionally, in some embodiments the N-terminal amino acid of thepolypeptide may be derivatized prior to conjugating the probe to theN-terminal amino acid. For example, in one embodiment the N-terminalamino acid is derivatized with an Edman reagent such as phenylisothiocyanate (PITC).

In one embodiment, the methods described herein include cleaving theN-terminal amino acid or N-terminal amino acid derivative of thepolypeptide using Edman, or related, chemical degradation. In oneembodiment, the methods described herein include cleaving the N-terminalamino acid or N-terminal amino acid derivative enzymatically with aprotease, for example an aminopeptidase.

In one embodiment, the methods described herein include comparing thespectral properties of the probe bound to an N-terminal amino acid of apolypeptide to a plurality of reference spectral properties. In oneembodiment, each reference spectral property is representative of theprobe conjugated to a different N-terminal amino acid. In oneembodiment, comparing the spectral properties of the probe to theplurality of reference spectral properties comprises comparing thespectra of the probe bound to the N-terminal amino acid to a pluralityof reference spectra. In one embodiment, the reference spectra arespectra of the probe bound to known N-terminal amino acids, such as thespectra shown in FIG. 5. In one embodiment, the method comprisesidentifying the closest match between the spectra of the probe and thereference spectra, thereby identifying the N-terminal amino acid of thepolypeptide.

Various statistical methods known in the art may be used to compare thespectra of the probe and reference spectra in order to identify theclosest match and the N-terminal amino acid of the polypeptide.

In one embodiment, suitable methods generate a quantitative measure ofsimilarity or difference between the spectra and the reference spectra.In one embodiment, the methods described herein further comprisesgenerating a statistical measure or probability score that a spectra isindicative of the presence of a particular N-terminal amino acid residueconjugated to the probe. In one embodiment, the methods used herein forcomparing the spectral properties of an N-terminal amino acid-probeconjugate and a reference/control conjugate use one or moreprobabilistic algorithms. For example, a probabilistic algorithm can betrained to identify different N-terminal amino acids conjugated toHMRG-BOC using the spectral data provided in FIG. 5 associating specificspectra with specific N-terminal amino acids. Additional reference datasets suitable for training probabilistic algorithms can also begenerated using other probes that exhibit different spectral propertieswhen conjugated to different N-terminal amino acids. In one embodiment,machine learning, genetic algorithms, or principle component analysis(PCA) may be used for comparing spectra and reference spectra.

In another aspect, the methods and reagents described herein are usefulfor labeling and sequencing a plurality of polypeptides in parallel. Forexample, in one embodiment the methods described herein includeconjugating a plurality of probe molecules to the N-terminal amino acidof each of the plurality of polypeptide molecules. In one embodiment,the method comprises detecting one or more spectral properties for eachprobe conjugated to the N-terminal amino acid of each of the pluralityof polypeptides. The N-terminal amino acid of each of the plurality ofpolypeptides can then be identified by comparing the plurality ofspectra properties to a plurality of reference (standards) spectralproperties.

In one embodiment, the method comprises detecting one or more spectralproperties for each probe conjugated to the N-terminal amino acid ofeach of the plurality of polypeptides at spatially resolved locations ina sample.

Different techniques known in the art may be used to detect spectralproperties of different molecules at spatially resolved locations. Forexample, super resolution microscopy may be used to detect one or morespectral properties of a probe conjugated to the N-terminal amino acidat a particular location within a sample. In one embodiment, the methodsdescribed herein use stochastic optical reconstruction microscopy(STORM).

In one embodiment, the detecting the spectra properties of a probeincludes ultrasensitive detection systems that are able to repeatedlydetect signals from precisely the same co-ordinates in a sample, therebyassigning the detected spectral information to a unique polypeptidemolecule.

In one embodiment, the spectral properties are detected using an opticaldetection system. Optical detection systems include a charge-coupleddevice (CCD), electron multiplying CCD (EMCCD), near-field scanningmicroscopy, far-field confocal microscopy, wide-field epi-illumination,light scattering, dark field microscopy, photoconversion, single and/ormultiphoton excitation, spectral wavelength discrimination, fluorophoreidentification, evanescent wave illumination, total internal reflectionfluorescence (TIRF) microscopy, super-resolution fluorescencemicroscopy, single-molecule localization microscopy, and single-moleculespectroscopy. In general, methods involve detection of laser-activatedfluorescence using a microscope equipped with a camera, sometimesreferred to as high-efficiency photon detection system. Suitable photondetection systems include, but are not limited to, photodiodes andintensified CCD cameras.

In one embodiment, examples of techniques suitable for single moleculedetection of the spectral properties of probes include confocal laser(scanning) microscopy, wide-field microscopy, near-field microscopy,fluorescence lifetime imaging microscopy, fluorescence correlationspectroscopy, fluorescence intensity distribution analysis, measuringbrightness changes induced by quenching/dequenching of fluorescence, orfluorescence energy transfer.

In a further aspect of the disclosure, the N-terminal amino acid of thepolypeptide is cleaved. Cleaving exposes the N-terminal amino group ofan adjacent (penultimate) amino acid on the polypeptide, whereby theadjacent amino acid is available for reaction with a new probe.Optionally, the polypeptide is sequentially cleaved until the last aminoacid in the polypeptide (C-terminal amino acid).

In one embodiment, sequential chemical degradation is used to cleave theN-terminal amino acid of the polypeptide. Edman degradation generallycomprises two steps, a coupling step and a cleaving step. These stepsmay be iteratively repeated, each time removing the exposed N-terminalamino acid residue of a polypeptide. In one embodiment Edman degradationproceeds by way of contacting the polypeptide with a suitable Edmanreagent such as PITC, or a ITC-containing analogue, at an elevated pH toform a N-terminal thiocarbamyl derivative. Reducing the pH, such by theaddition of trifluoroacetic acid results in the cleaving the N-terminalamino acid thiocarbamyl derivative from the polypeptide to form a freeanilinothiozolinone (ATZ) derivative. Optionally, this ATZ derivativemay be washed away from the sample. In one embodiment the pH of thesample is modulated in order to control the reactions governing thecoupling and cleaving steps.

In some embodiments, the N-terminal amino acid is contacted with asuitable Edman reagent such as PITC, or an ITC containing analogue, atan elevated pH prior to contacting the affixed polypeptide with aplurality of probes that selectively bind the N-terminal amino acidderivative. Optionally, the cleaving step comprises reducing the pH inorder to cleave the N-terminal amino acid derivative.

In one embodiment of the description, the method includes comparing thesequence obtained for each polypeptide molecule to a reference proteinsequence database. In some embodiments, small fragments comprising10-20, or fewer, sequenced amino acid residues, consecutive or withgaps, may be useful for detecting the identity of a polypeptide in asample.

The following examples illustrate embodiments of the invention and arenot intended to limit the scope of the invention.

Example 1: Single Molecule Spectroscopy-Based Amino Acid ResidueIdentification

The inventor has adapted super-resolution fluorescence microscopy to anEdman-like sequencing process, allowing for the simultaneous, massivelyparallel identification and counting of large numbers of individualprotein molecules. The protein molecules may be in vitro or in situ suchas in cells and/or tissues. N-terminal amino acid residues of affixedpolypeptides are reacted with a fluorescent probe that confers distinctspectral properties upon coupling to different amino acids. Theresulting characteristic emission profile generated by the distinctN-terminal derivative formed on individual protein molecules ismonitored by super-resolution spectroscopy to determine the identity ofthe corresponding cognate amino acid residue, which is then cleaved off,such as through Edman-like chemistry. Through multiple, iterative cyclicrounds of coupling the remaining polypeptide portions to fresh dye,re-imaging the population of N-terminal probes, followed by selectivecleavage of consecutive probe derivatives, partial sequence and preciselocalization information is obtained for thousands to millions ofprotein molecules imaged in a concurrent manner. Given access to genomicinformation, the probe used for imaging need not resolve all of theamino acid side chains, but rather just a sufficient subset, in seriesor with gaps, in order to unambiguously identify all of the proteinmolecules present in a biological sample.

Fluorescence refers to the ability of certain molecules, such as organicdyes, to absorb light at a particular wavelength and, after a briefinterval, emit light at a different (longer) wavelength. Fluorescenceproperties that can be precisely measured include emission intensity,polarity/anisotropy, and lifetime. To detect a particular proteintarget, fluorophores are usually covalently coupled to an antibody. Dueto diffraction of the dye emission wave, the smallest features normallyresolvable by microscopy are ˜250 nm in the lateral (x-y) plane.Overlapping concurrent emissions from adjacent probes usually obscuressmaller features, preventing determination of individual componentspresent in structures like the cell membrane, nucleus or cytoskeleton,or multiprotein complexes. However, super-resolution imaging techniques,allow for the precise localization of individual fluorescently labeledprotein molecules. Methods like STORM achieve sub-diffraction resolutionby spatially and temporally separating the fluorescence emission ofindividual fluorophores through reversible, stochastic transitioning ofonly a small fraction from a dark (off) state to a bright (on) state,such that only one molecule is detected per diffraction-limited area.Using ultrasensitive digital cameras to detect these transient lowintensity signals at high speed, the imaging process is repeated untilall probes present in a field of view are detected sequentially,typically over 10,000+ frames that are each populated with a sparsesubset of dye emissions. Individual molecules are then preciselylocalized using software to fit centroids over each signal, from which afinal super-resolution image is reconstructed. While compatible withlive cell or 3D imaging, single molecule imaging currently requireshighly selective probes (e.g. antibodies), and only limited targetmultiplexing (simultaneous detection of different proteins) has beenachieved.

The inventor has determined that characteristic changes in theproperties of certain fluorescent dyes occur when covalently bound todifferent amino acids.

For example, high yield xanthene dyes that predominate as darkspirocyclic structures (eg. rhodamine lactone) but reversibly open torestore fluorescence are ideally suited to single molecule sequencing.Photoswitching is usually controlled by experimental conditions (eg.using thiols, irradiation intensity, pH), but Uno et al. (Nat Chem. 2014August; 6(8):681-9) recently reported xanthene-based dyes that blinkspontaneously.

As shown in FIG. 1 by synthesizing a reactive isothiocyanate group ontoa blinking dye (to confer Edman-like degradation), the inventor hasdetermined that it is possible to (i) efficiently label the N-terminalsof individual polypeptide molecules, (ii) detect fluorescent emissionand blinking by single molecule imaging, and (iii) selectively cleaveoff the conjugated N-terminal residue, revealing the next (penultimate)amino acid. The inventor also determined that since the emission profile(intensity, wavelength shape, other spectral properties) of some dyes isinfluenced by the local chemical (electron donating/extracting)environment, small, but characteristic, changes in emission properties(such as a reproducible shift in wavelength profile) formed afterconjugation to different amino acids can be used to infer the identityof the corresponding labeled N-terminal residue. By repeating thelabeling, imaging, and degradation process in an iterative manner, theidentity (partial sequence) and abundance (occurrences) of large numbersof individual protein molecules may be determined simultaneously.

These methods and techniques may be used for the unbiased sequencing andcounting of polypeptides, present either on a slide or flow cell, or atthe plasma membrane or within intracellular organelles of a cell,thereby revealing the composition, localization and physicalinteractions of proteins that are present.

After covalent coupling of the probe to the N-terminal, an “opticalsignature” unique to one or more particular cognate amino acids isidentified. The underlying rationale is that atomic interactions,differentially induced by adjacent amino acid side chains, affect theelectronic states and hence ground and excited states of the dyedifferently, leading to subtle, but reproducible, variations in emissionintensity, polarity, or peak shape. The spectral profiles of individualfluorescent dye molecules using a wide-field imaging method (forexample, spectrally resolved stochastic optical reconstructionmicroscopy, SR-STORM; see e.g. Zhang et al. Nat Methods. 2015 October;12(10):935-8)) which can deconvolute small shifts in emission intensityor peak shape.

Remarkably, as shown in FIG. 2, the emission of certainrhodamine-derivatives is reproducibly altered in solution upon couplingto different amino acids (λ^(emm) ˜535 nm for Met vs. 524 nm for Trp).

Example 2: Identification and Characterization of Fluorescent Probes

Using fluorescent probes some, or all, of the 20+ naturally occurringamino acids may be distinguished by the detecting the fluorescentemission of the individual probes conjugated to different amino acids.Various dye molecules, such HMRG and other xanthene-derivatives, areinvestigated for their suitability as probes for single moleculespectroscopy-based residue identification.

Experiments are conducted in order to identify fluorescent probessuitable for distinguishing different amino acids and to define optimalimaging conditions (eg. buffer pH/polarity) that maximizediscrimination, as well as the influence, if any, of adjacent aminoacids (eg. penultimate residue). Large numbers of individual probemolecules are examined after immobilization to a surface (eg. coverslip,flowcell, or microbead), or natively in/on metazoan cells, microbes orvirus, in order to derive a precise, imaging-based N-terminal readoutfor one, or more, optically-encoded fluorescent probes. To allow foriterative sequencing, the probe(s) may support N-terminal cleavage, suchas through isothiocyanate-mediated degradation.

The surface of borosilicate coverglass is covered with proteins, eitherin a folded or denatured state, over a serially diluted concentrationrange (down to sub-ELISA detection limits). Analytes include syntheticpeptides, polypeptides, and/or recombinant proteins, includingantibodies, receptors, toxins, or enzymes; negative controls (no targetor dye) are likewise assessed in parallel. Prior to exposure to theprobe, reactive lysine side chains are chemically modified. To overcomenative N-terminal blocks (eg. acetylation), which preclude sequencedetermination, proteases may be used to liberate a free amino group.After coupling to the probe, imaging is performed on a STORM-capableinverted total reflection fluorescence microscope, sampling multiplefields of view for statistical analysis. At 90× magnification, thesurface area in each field of view is ˜3600 μm², allowing hundreds ofthousands of molecules to be imaged simultaneously.

For image analysis, ImageJ (available online from the NationalInstitutes of Health, Bethesda, Md.) and other digital image processingsoftware is used to process the image stacks of recorded probe emissionsto identify individual fluorophores that blink and vanish. Aftercorrecting for lateral drift, probe locations (reporter coordinates andestimated uncertainty in nanometers) and differences in intensity andwavelength, or other emission properties, are calculated withsub-diffraction precision. Each optically-encoded reporter isindividually classified by matching the emission profile to reference(standard) spectra to identify the cognate N-terminal amino acid.Spectral properties such as peak shape (maxima) and intensity, asfluorescent polarization/anisotropy and lifetimes will be measured. Theprobes are compared against well characterized antibodies labeled withstandard STORM dyes (eg. Alexa647) in order to demonstrate highlyselective target discrimination with exceptional quantitative accuracyand sensitivity.

Example 3: HMRG-BOC-ITC Coupling and Identifying Different N-TerminalAmino Acid by Fluorescence Profiling Materials and Methods

Hydroxymethyl rhodamine green (HMRG) tert-butyloxycarbonyl (BOC)isothiocyanate (ITC) (HMRG-BOC-ITC) as shown in FIG. 4A was synthesizedas shown in FIG. 4B and dissolved in DMSO to a final concentration 34nmol/μl.

Peptide beads: Peptide beads were synthesized by Kinexus (VancouverB.C). Tentagel resin (Tenta-Gel® M NH2, 10 μm beads, RAPP Polymer) wasused as the support for solid peptide synthesis. The peptides weresynthesized to have the amino acid sequence X-AGWYMRLG (SEQ ID NO: 1),where X represents any one of 20 different amino acids at N-termini ofthe peptide. Approximately half (˜50%) of each peptide/resin contained acleavable HMBA linker inserted between peptide sequence and the beadwhich enabled cleavage of the peptide molecules from the beads upontreatment with base.

Prior to coupling, 100 μl of dimethylformamide (DMF) was added to eachtube containing dry peptide resin (˜1.5-2 μmole of peptide). The beadslurry was stored at −20° C. and used as needed for coupling.

The coupling reaction contained the following:

5 μl of peptide bead slurry (75-100 nmol of peptide in DMF);

3.8 μl of HMRG-BOC-ITC in DMSO (129 nmol);

8.3 μl pyridine; and

3.6 μl dichloromethane (DCM).

As a non-peptide control, uncoupled tentagel resin not containing anypeptide was treated with 2% acetic anhydride and 2%N,N-Diisopropylethylamine in DMF for at least 5 min in order to blockfree reactive NH2-groups. Solvents were evaporated and beads resuspendedin DMF and used for coupling.

The coupling reaction was performed as follows. 5 μl of each of thepeptide-bead slurry (75-100 nmol of peptide in DMF) and theblocked-beads slurry as a negative control (see below) was aliquotedinto separate microcentrifuge tubes. A master mix containing 3.8 μl ofdye HMRG-BOC-ITC (34 nmol/μl), 8.3 μl of pyridine and 3.6 μl ofdichloromethane (DCM) was added to each tube. The tubes were incubatedwith gentle shaking in the dark overnight at 4° C.

For the non-peptide control, Tentagel resin (not containing any peptide)was treated with 2% acetic anhydride and 2% N,N-Diisopropylethylamine inDMF for at least 5 min in order to block free NH2-groups. Solvents wereevaporated and the beads were resuspended in DMF and used as a control.

The next day coupled beads were washed extensively to remove uncoupleddye: 200 μl of DCM was added to each tube and the mixture transferred to0.45 μm spin filter (UFC30HVNB, Amicon). After a short 30 second spin at6000 rpm the flow-through was discarded and the beads retained on thefilter were washed with the following solvents: DCM (2×),THF(tetrahydrofuran)(2×), and THF (20%)(4×), followed by short spins anddiscarding the flow-through. After washing, dry peptide resin wasresuspended in 150 μl of 30% NH₄OH and transferred to a fresh tube.Tubes were incubated with gentle shaking at room temperature. After anincubation of 1-2 hours, the bead suspension (in NH₄OH) was transferredback to a spin filter and briefly centrifuged. The flowthrough,containing free cleaved-off peptides, was collected into a fresh tubeand vacuum dried to remove the base. Dried peptides were resuspended in150-200 μl of either of 10 mM phosphate buffer, pH7.4 containing 5%DMSO, or same buffer with 10% THF.

Fluorescence spectroscopic data of individual HMRG-BOC-coupled peptideswas collected on a fluorometer (Fluorolog, Horiba) using 480 nmexcitation light and emission profiles were acquired from 510 to 550 nm.The slit width of excitation and emission was 5 nm.

Results

The normalized intensity for each of the 20 different N-terminalconjugated HMRG-BOC-coupled peptides tested is shown in FIG. 5. Thespectra exhibited different spectral signatures allowing for differentN-terminal amino acids to be distinguished. For example, HMRG-BOCcoupled to aspartic acid (D) exhibited a lower normalized intensity athigher wavelengths (540-550 nm) relative to other amino acids. HMRG-BOCcoupled to N-terminal lysine (K) exhibited a lower normalized intensityat lower wavelengths (510-520 nm) relative to the other amino acids.HMRG-BOC coupled to N-terminal Asparagine (N) exhibited a distinctiveincrease in normalized intensity going from 540 nm to 550 nm.

Analysis of the spectra shown in FIG. 5 also indicated certain generaltrends in the spectral properties of the N-terminal amino acid-probeconjugates. As shown in FIG. 6, N-terminal amino acids that arehydrophobic such as Leucine tended to exhibit a red-shift in intensityrelative to the average. N-terminal amino acids that are hydrophilicsuch as Aspartic acid tended to exhibit a blue-shift in intensityrelative to the average.

By sequentially conjugating a fluorescent probe such as HMRG-BOC-ITC tothe N-terminal amino acid of a polypeptide, obtaining a spectra of theprobe-polypeptide conjugate, identifying the N-terminal amino acid ofthe polypeptide based on comparing the spectra to one or more referencespectra such as those shown in FIG. 5, and the cleaving the N-terminalamino acid of the polypeptide the sequence of the polypeptide may bedetermined.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or adaptations of the invention following, in general, theprinciples of the invention and including such departures from thepresent disclosures as come within known or customary practice withinthe art to which the invention pertains and as may be applied to theessential features herein before set forth, and as follows in the scopeof the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

REFERENCES

-   Uno et al. A spontaneously blinking fluorophore based on    intramolecular spirocyclization for live-cell super-resolution    imaging. Nat Chem. 2014 August; 6(8):681-9-   Zhang et al. Ultrahigh-throughput single-molecule spectroscopy and    spectrally resolved super-resolution microscopy. Nature Methods 12,    935-938 (2015).

1. A method of sequencing a polypeptide comprising: a) conjugating aprobe to an N-terminal amino acid of the polypeptide wherein the probeexhibits different spectral properties when conjugated to differentN-terminal amino acids; b) detecting one or more spectral properties ofthe probe conjugated to the N-terminal amino acid; c) identifying thecorresponding N-terminal amino acid of the polypeptide by comparing thespectral properties of the probe to a plurality of reference spectralproperties, wherein each reference spectral property is representativeof the probe conjugated to a different N-terminal amino acid; d)cleaving the N-terminal amino acid of the polypeptide; and e) repeatingsteps (a) to (d) to determine the sequence of at least a portion of thepolypeptide.
 2. The method of claim 1, wherein the polypeptide is asingle polypeptide molecule.
 3. The method of claim 1, wherein the probeis covalently conjugated to the N-terminal amino acid of thepolypeptide.
 4. The method of claim 1, wherein detecting one or morespectral properties comprises detecting fluorescence of the probe boundto the N-terminal amino acid of the polypeptide.
 5. The method of claim1, wherein detecting one or more spectral properties comprises detectingfluorescence emission intensity, polarity/anisotropy or lifetime.
 6. Themethod of claim 5, comprising detecting the emission intensity,polarity/anisotropy or lifetime at a single wavelength or at a pluralityof wavelengths.
 7. (canceled)
 8. The method of any one of claim 1,wherein the probe comprises a xanthene derivative.
 9. The method ofclaim 8, wherein the xanthene derivative is based on fluorescein, eosin,or rhodamine.
 10. The method of claim 9, wherein the probe comprises aderivative of hydroxymethyl rhodamine green (HMRG).
 11. The method ofclaim 1, wherein the probe comprises a spontaneously blinking dye or aphotoswitchable dye.
 12. The method of claim 1, wherein the probecomprises a reactive/labile isothiocyanate group.
 13. The method ofclaim 12, wherein step d) comprises cleaving the N-terminal amino acidor N-terminal amino acid derivative of the polypeptide using Edmanchemical degradation.
 14. (canceled)
 15. (canceled)
 16. The method ofclaim 1, comprising sequencing a plurality of polypeptides in parallelwherein step a) comprises conjugating a plurality of probes to theN-terminal amino acid of each of the plurality of polypeptides, step b)comprises detecting one or more spectral properties for each probeconjugated to the N-terminal amino acid of each of the plurality ofpolypeptides and step c) comprises identifying the N-terminal amino acidof each of the plurality of polypeptides by comparing the plurality ofspectra properties to a plurality of standard spectral properties. 17.The method of claim 16, wherein the method comprises detecting one ormore spectral properties for each probe conjugated to the N-terminalamino acid of each of the plurality of polypeptides at spatiallyresolved locations in a sample containing the plurality of polypeptides.18. The method of claim 1, wherein detecting one or more spectralproperties of the probe conjugated to the N-terminal amino acidcomprises super resolution microscopy.
 19. The method of claim 18,wherein the super resolution microscopy comprises stochastic opticalreconstruction microscopy (STORM).
 20. The method of claim 1, furthercomprising comparing the sequence of at least one polypeptide moleculedetermined in step e) to a reference protein sequence database.
 21. Themethod of claim 1, wherein comparing the spectral properties of theprobe to a plurality of reference spectral properties comprises the useof machine learning, genetic algorithms, or principle component analysis(PCA).
 22. The method of claim 1, wherein step b) comprises obtaining afluorescence emission spectra of the probe conjugated to the N-terminalamino acid and step c) comprising comparing the fluorescence emissionspectra to a plurality of reference spectra, wherein each referencespectra is representative of the probe conjugated to a differentN-terminal amino acid.
 23. The method of claim 1, wherein the C-terminalend of the polypeptide is conjugated to a substrate, optionally througha linker. 24.-26. (canceled)